Dahlquist:DNA Microarray Protocol - Revision history

Kam D. Dahlquist: /* Day 4 */ small edit to protocol;

Kam D. Dahlquist — Wed, 27 Jun 2012 22:41:41 GMT

Day 4: small edit to protocol;

Kam D. Dahlquist: /* Day 1 */ revised to new Ambion protocol

Kam D. Dahlquist — Wed, 27 Jun 2012 22:21:15 GMT

Day 1: revised to new Ambion protocol

←Older revision		Revision as of 22:21, 27 June 2012
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	===Day 1===		===Day 1===
		+
		+	'''''Note that Ambion/Applied Biosystems/Life Technologies revised the protocol for the AM1753 Amino Allyl MessageAmp II aRNA Amplification Kit and we started using the revised protocol on 12/9/10.'''''

	# Dry 1 μg of yeast total RNA to less than 10 μL in a SpeedVac.		# Dry 1 μg of yeast total RNA to less than 10 μL in a SpeedVac.
	#* 1 μg is the largest amount recommended for the kit.		#* 1 μg is the largest amount recommended for the kit.
	#* RNA can be dried ahead of time and stored at -80°C.		#* RNA can be dried ahead of time and stored at -80°C.
-	# '''Reverse Transcription to Synthesize First Strand of cDNA:''' follow steps C1-C4 in protocol.	+	# '''Reverse Transcription to Synthesize First Strand of cDNA:''' follow steps C1-C3 in protocol.
-	~~#* Can use heat block for 70°C incubation in step C2~~.	+
	#* Use 42°C water bath for step C4.		#* Use 42°C water bath for step C4.
	#* Thaw reagents and make Master Mix for step D1 and immediately proceed to step D1 when 2 hours are up.		#* Thaw reagents and make Master Mix for step D1 and immediately proceed to step D1 when 2 hours are up.
Line 114:		Line 115:
	#* Use 16°C water bath in the cold room for step D2.		#* Use 16°C water bath in the cold room for step D2.
	#* Potential stopping point for Day 1. Freeze samples at -20°C. Protocol says it is better to go on to cDNA purification before freezing, though.		#* Potential stopping point for Day 1. Freeze samples at -20°C. Protocol says it is better to go on to cDNA purification before freezing, though.
-	# '''cDNA Purification:''' Aliquot nuclease-free water needed for elution step (E4) plus some extra into a 1.5 mL microcentrifuge tube and pre-heat to ~~50°C~~ in a water bath (the protocol says to heat the entire bottle, but this is unwieldy).	+	# '''cDNA Purification:''' Aliquot nuclease-free water needed for elution step (E4) plus some extra into a 1.5 mL microcentrifuge tube and pre-heat to 55°C in a water bath (the protocol says to heat the entire bottle, but this is unwieldy).
-	# Perform steps E1-E4.	+	# Perform steps E1-E5.
	# Stopping point for Day 1. Freeze samples at -20°C.		# Stopping point for Day 1. Freeze samples at -20°C.

Kam D. Dahlquist: /* Day 4 */ changed spin to 3 min at 5000 X g

Kam D. Dahlquist — Sat, 17 Mar 2012 21:44:01 GMT

Day 4: changed spin to 3 min at 5000 X g

←Older revision		Revision as of 21:44, 17 March 2012
Line 193:		Line 193:
	# Let sit at room temperature for 2 minutes, then spin for 3 minutes at 5,000 X g.		# Let sit at room temperature for 2 minutes, then spin for 3 minutes at 5,000 X g.
	# Repeat the elution with another 15 μL of nuclease-free water pre-heated to 50-60°C.		# Repeat the elution with another 15 μL of nuclease-free water pre-heated to 50-60°C.
-	# Let sit at room temperature for 2 minutes, then spin for ~~1.5~~ minutes at 10,000 X g.	+	# Let sit at room temperature for 2 minutes, then spin for 3 minutes at 5,000 X g.

	'''Determine the concentration of labeled aRNA'''		'''Determine the concentration of labeled aRNA'''

Kam D. Dahlquist: /* Slide Coating Solution */ added note that PEG is hygroscopic

Kam D. Dahlquist — Tue, 25 Jan 2011 23:53:55 GMT

Slide Coating Solution: added note that PEG is hygroscopic

←Older revision		Revision as of 23:53, 25 January 2011
Line 74:		Line 74:
	* Can be re-used (but not indefinitely)		* Can be re-used (but not indefinitely)
	* NOTE: PEG 8000 does '''NOT''' work in this recipe. It will not dissolve in the acetone. I have not tried MW cut-offs between 2000 and 8000, though. ''— [[User:Kam D. Dahlquist\|Kam D. Dahlquist]] 12:36, 22 July 2010 (EDT)''		* NOTE: PEG 8000 does '''NOT''' work in this recipe. It will not dissolve in the acetone. I have not tried MW cut-offs between 2000 and 8000, though. ''— [[User:Kam D. Dahlquist\|Kam D. Dahlquist]] 12:36, 22 July 2010 (EDT)''
		+	* NOTE: PEG is hygroscopic, store aliquots in dessicator. ''— [[User:Kam D. Dahlquist\|Kam D. Dahlquist]] 18:53, 25 January 2011 (EST)''

	===Equipment===		===Equipment===

Kam D. Dahlquist: /* Day 4 */ fixed formatting

Kam D. Dahlquist — Wed, 13 Oct 2010 20:58:39 GMT

Day 4: fixed formatting

←Older revision		Revision as of 20:58, 13 October 2010
Line 183:		Line 183:
	# Add 105 μL of aRNA Binding Buffer to each labeled aRNA sample--proceed to the next step immediately.		# Add 105 μL of aRNA Binding Buffer to each labeled aRNA sample--proceed to the next step immediately.
	# Add 75 μL of 100% ethanol to each sample, and mix by pipetting the mixture up and down 3 times. '''''Do NOT vortex and do NOT centrifuge.'''''		# Add 75 μL of 100% ethanol to each sample, and mix by pipetting the mixture up and down 3 times. '''''Do NOT vortex and do NOT centrifuge.'''''
-	# Place aRNA filter cartridges into the appropriate number of collection tubes and pipet each sample from the previous step onto the appropriate column. <s>~~Spin~~ 1 minute at 10,000 X g</s>. Discard flow-through.	+	# Place aRNA filter cartridges into the appropriate number of collection tubes and pipet each sample from the previous step onto the appropriate column. Spin <s>1 minute at 10,000 X g</s> 2 minutes at 5,000 X g. Discard flow-through.
-	#* ''Note that we started having issues with the spin columns when Ambion/Applied Biosystems got purchased by Invitrogen and they started supplying Invitrogen columns in the kit. The resin would come down into the collection tube. We now spin for 2 minutes at ~~5000~~ X g instead.''	+	#* ''Note that we started having issues with the spin columns when Ambion/Applied Biosystems got purchased by Invitrogen and they started supplying Invitrogen columns in the kit. The resin would come down into the collection tube. We now spin for 2 minutes at 5,000 X g instead.''
	# Apply 500 μL Wash Buffer to each filter cartridge. Spin 2 minutes at 5,000 X g. Discard flow-through.		# Apply 500 μL Wash Buffer to each filter cartridge. Spin 2 minutes at 5,000 X g. Discard flow-through.
	# Spin again at 5,000 X g to remove trace amounts of Wash Buffer.		# Spin again at 5,000 X g to remove trace amounts of Wash Buffer.

Kam D. Dahlquist: /* Day 4 */ revised spins

Kam D. Dahlquist — Wed, 13 Oct 2010 20:57:43 GMT

Day 4: revised spins

←Older revision		Revision as of 20:57, 13 October 2010
Line 183:		Line 183:
	# Add 105 μL of aRNA Binding Buffer to each labeled aRNA sample--proceed to the next step immediately.		# Add 105 μL of aRNA Binding Buffer to each labeled aRNA sample--proceed to the next step immediately.
	# Add 75 μL of 100% ethanol to each sample, and mix by pipetting the mixture up and down 3 times. '''''Do NOT vortex and do NOT centrifuge.'''''		# Add 75 μL of 100% ethanol to each sample, and mix by pipetting the mixture up and down 3 times. '''''Do NOT vortex and do NOT centrifuge.'''''
-	# Place aRNA filter cartridges into the appropriate number of collection tubes and pipet each sample from the previous step onto the appropriate column. Spin 1 minute at 10,000 X g. Discard flow-through.	+	# Place aRNA filter cartridges into the appropriate number of collection tubes and pipet each sample from the previous step onto the appropriate column. <s>Spin 1 minute at 10,000 X g</s>. Discard flow-through.
-	# Apply 500 μL Wash Buffer to each filter cartridge. Spin ~~1 minute~~ at 10,000 X g. Discard flow-through.	+	#* ''Note that we started having issues with the spin columns when Ambion/Applied Biosystems got purchased by Invitrogen and they started supplying Invitrogen columns in the kit. The resin would come down into the collection tube. We now spin for 2 minutes at 5000 X g instead.''
-	# Spin again at 10,000 X g to remove trace amounts of Wash Buffer.	+	# Apply 500 μL Wash Buffer to each filter cartridge. Spin 2 minutes at 5,000 X g. Discard flow-through.
		+	# Spin again at 5,000 X g to remove trace amounts of Wash Buffer.
	# Transfer filter cartridge into a fresh collection tube, being careful not to contaminate the new tube with any of the flow through from the previous step.		# Transfer filter cartridge into a fresh collection tube, being careful not to contaminate the new tube with any of the flow through from the previous step.
	# To the center of the filter, add 15 μL of the nuclease-free water that you pre-heated to 50-60°C in step 1.		# To the center of the filter, add 15 μL of the nuclease-free water that you pre-heated to 50-60°C in step 1.
	#* Note: the manufacturer's protocol calls for eluting twice with ''10 μL'' of the nuclease-free water, not 15 μL. However, we have observed that 5-8 μL of the total elution volume is retained on the column. By increasing the volumn slightly for each elution step we have increased our yield of labeled aRNA without diluting it too much. ''— [[User:Kam D. Dahlquist\|Kam D. Dahlquist]] 12:47, 22 July 2010 (EDT)''		#* Note: the manufacturer's protocol calls for eluting twice with ''10 μL'' of the nuclease-free water, not 15 μL. However, we have observed that 5-8 μL of the total elution volume is retained on the column. By increasing the volumn slightly for each elution step we have increased our yield of labeled aRNA without diluting it too much. ''— [[User:Kam D. Dahlquist\|Kam D. Dahlquist]] 12:47, 22 July 2010 (EDT)''
-	# Let sit at room temperature for 2 minutes, then spin for ~~1.5~~ minutes at 10,000 X g.	+	# Let sit at room temperature for 2 minutes, then spin for 3 minutes at 5,000 X g.
	# Repeat the elution with another 15 μL of nuclease-free water pre-heated to 50-60°C.		# Repeat the elution with another 15 μL of nuclease-free water pre-heated to 50-60°C.
	# Let sit at room temperature for 2 minutes, then spin for 1.5 minutes at 10,000 X g.		# Let sit at room temperature for 2 minutes, then spin for 1.5 minutes at 10,000 X g.

Kam D. Dahlquist: /* Slide Coating Solution / removed extra

Kam D. Dahlquist — Tue, 12 Oct 2010 16:50:37 GMT

Slide Coating Solution: removed extra *

←Older revision		Revision as of 16:50, 12 October 2010
Line 68:		Line 68:

	==== Slide Coating Solution ====		==== Slide Coating Solution ====
-	** "Home-brew" version of Genisphere DyeSaver 2	+	* "Home-brew" version of Genisphere DyeSaver 2
-	** 2% PEG (MW cut-off 2000) in 1:1 solution of acetone and toluene	+	* 2% PEG (MW cut-off 2000) in 1:1 solution of acetone and toluene
-	** 200 mL needed to fill Coplin jar	+	* 200 mL needed to fill Coplin jar
-	** Weigh 4.0 g of polyethylene glycol (Mn ca. 2000) and transfer to a dry glass reagent bottle. Add 100 mL of HPLC grade acetone (water content less than 0.5%) and close the bottle with a lid. Swirl the bottle gently to dissolve the solid completely. When a clear solution is formed add 100 mL of anhydrous toluene and mix the solution before using in the coating experiments. Note: do not use any plastic in this procedure. Store at room temperature in a fume hood.	+	* Weigh 4.0 g of polyethylene glycol (Mn ca. 2000) and transfer to a dry glass reagent bottle. Add 100 mL of HPLC grade acetone (water content less than 0.5%) and close the bottle with a lid. Swirl the bottle gently to dissolve the solid completely. When a clear solution is formed add 100 mL of anhydrous toluene and mix the solution before using in the coating experiments. Note: do not use any plastic in this procedure. Store at room temperature in a fume hood.
-	** Can be re-used (but not indefinitely)	+	* Can be re-used (but not indefinitely)
-	** NOTE: PEG 8000 does '''NOT''' work in this recipe. It will not dissolve in the acetone. I have not tried MW cut-offs between 2000 and 8000, though. ''— [[User:Kam D. Dahlquist\|Kam D. Dahlquist]] 12:36, 22 July 2010 (EDT)''	+	* NOTE: PEG 8000 does '''NOT''' work in this recipe. It will not dissolve in the acetone. I have not tried MW cut-offs between 2000 and 8000, though. ''— [[User:Kam D. Dahlquist\|Kam D. Dahlquist]] 12:36, 22 July 2010 (EDT)''

	===Equipment===		===Equipment===

Kam D. Dahlquist: /* Reagents */ removed empty line

Kam D. Dahlquist — Tue, 12 Oct 2010 16:50:11 GMT

Reagents: removed empty line

←Older revision		Revision as of 16:50, 12 October 2010
Line 66:		Line 66:
	* Yeast 6.4K Array (Y6.4K) ([http://www.microarrays.ca/products/types.html#Y6.4K University Health Network Microarray Centre, Toronto])		* Yeast 6.4K Array (Y6.4K) ([http://www.microarrays.ca/products/types.html#Y6.4K University Health Network Microarray Centre, Toronto])
	** NOTE: they have discontinued printing these chips as of 7/31/10. ''— [[User:Kam D. Dahlquist\|Kam D. Dahlquist]] 12:38, 22 July 2010 (EDT)''		** NOTE: they have discontinued printing these chips as of 7/31/10. ''— [[User:Kam D. Dahlquist\|Kam D. Dahlquist]] 12:38, 22 July 2010 (EDT)''
-

	==== Slide Coating Solution ====		==== Slide Coating Solution ====

Kam D. Dahlquist: /* Reagents */ split slide coating solution to own heading

Kam D. Dahlquist — Tue, 12 Oct 2010 16:49:53 GMT

Reagents: split slide coating solution to own heading

←Older revision		Revision as of 16:49, 12 October 2010
Line 66:		Line 66:
	* Yeast 6.4K Array (Y6.4K) ([http://www.microarrays.ca/products/types.html#Y6.4K University Health Network Microarray Centre, Toronto])		* Yeast 6.4K Array (Y6.4K) ([http://www.microarrays.ca/products/types.html#Y6.4K University Health Network Microarray Centre, Toronto])
	** NOTE: they have discontinued printing these chips as of 7/31/10. ''— [[User:Kam D. Dahlquist\|Kam D. Dahlquist]] 12:38, 22 July 2010 (EDT)''		** NOTE: they have discontinued printing these chips as of 7/31/10. ''— [[User:Kam D. Dahlquist\|Kam D. Dahlquist]] 12:38, 22 July 2010 (EDT)''
-	* Slide Coating Solution	+
		+
		+	==== Slide Coating Solution ====
	** "Home-brew" version of Genisphere DyeSaver 2		** "Home-brew" version of Genisphere DyeSaver 2
	** 2% PEG (MW cut-off 2000) in 1:1 solution of acetone and toluene		** 2% PEG (MW cut-off 2000) in 1:1 solution of acetone and toluene

Kam D. Dahlquist: /* Supplies */ note about spec

Kam D. Dahlquist — Thu, 22 Jul 2010 16:57:32 GMT

Supplies: note about spec

←Older revision		Revision as of 16:57, 22 July 2010
Line 25:		Line 25:
	** UV-transparant microcuvettes hold 50 μL total volume		** UV-transparant microcuvettes hold 50 μL total volume
	** can be cleaned and re-used indefinitely when you don't need to recover the sample from them		** can be cleaned and re-used indefinitely when you don't need to recover the sample from them
		+	** you don't need these if you have access to an [http://www.implen.com/nanophotometer.html Implen NanoPhotometer] (or Thermo NanoDrop or GE Nanovue)
	* RNaseZap (Denville Scientific Catalog <nowiki>#</nowiki>D1180)		* RNaseZap (Denville Scientific Catalog <nowiki>#</nowiki>D1180)
	** Actually an Applied Biosystems/Ambion product Catalog <nowiki>#</nowiki>AM9780, but it is less expensive if you buy it from Denville		** Actually an Applied Biosystems/Ambion product Catalog <nowiki>#</nowiki>AM9780, but it is less expensive if you buy it from Denville

←Older revision		Revision as of 22:41, 27 June 2012
Line 174:		Line 174:
	# To tube containing dried aRNA, add 9 μL Coupling Buffer and mix well/flash spin.		# To tube containing dried aRNA, add 9 μL Coupling Buffer and mix well/flash spin.
	# Resuspend each CyDye pack in 11 μL of DMSO [and keep in the dark at room temperature for up to 1 hour until ready to use it.] You will need one Cy3 and one Cy5 pack per microarray.		# Resuspend each CyDye pack in 11 μL of DMSO [and keep in the dark at room temperature for up to 1 hour until ready to use it.] You will need one Cy3 and one Cy5 pack per microarray.
		+	#* It is helpful to flash spin the dried dye before adding the DMSO to make sure it is at the bottom of the tube.
	# To tube containing the aRNA:Coupling Buffer mixture, add 11 µL of dye to each tube, mix thoroughly and flash spin.		# To tube containing the aRNA:Coupling Buffer mixture, add 11 µL of dye to each tube, mix thoroughly and flash spin.
	#* NOTE: Make sure you know which dye to add to which tube!		#* NOTE: Make sure you know which dye to add to which tube!
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	'''Removing Uncoupled Dye Material'''		'''Removing Uncoupled Dye Material'''

-	# Before beginning the procedure, pipet enough nuclease-free water into a fresh tube so that you have 30 μL X number of tubes, plus a little extra. Pre-heat to ~~50-60°C~~. The water should heat for at least 10 minutes.	+	# Before beginning the procedure, pipet enough nuclease-free water into a fresh tube so that you have 30 μL X number of tubes, plus a little extra. Pre-heat to 55°C. The water should heat for at least 10 minutes.
	# Add 105 μL of aRNA Binding Buffer to each labeled aRNA sample--proceed to the next step immediately.		# Add 105 μL of aRNA Binding Buffer to each labeled aRNA sample--proceed to the next step immediately.
	# Add 75 μL of 100% ethanol to each sample, and mix by pipetting the mixture up and down 3 times. '''''Do NOT vortex and do NOT centrifuge.'''''		# Add 75 μL of 100% ethanol to each sample, and mix by pipetting the mixture up and down 3 times. '''''Do NOT vortex and do NOT centrifuge.'''''
Line 190:		Line 191:
	# Spin again at 5,000 X g to remove trace amounts of Wash Buffer.		# Spin again at 5,000 X g to remove trace amounts of Wash Buffer.
	# Transfer filter cartridge into a fresh collection tube, being careful not to contaminate the new tube with any of the flow through from the previous step.		# Transfer filter cartridge into a fresh collection tube, being careful not to contaminate the new tube with any of the flow through from the previous step.
-	# To the center of the filter, add 15 μL of the nuclease-free water that you pre-heated to ~~50-60°C~~ in step 1.	+	# To the center of the filter, add 15 μL of the nuclease-free water that you pre-heated to 55°C in step 1.
	#* Note: the manufacturer's protocol calls for eluting twice with ''10 μL'' of the nuclease-free water, not 15 μL. However, we have observed that 5-8 μL of the total elution volume is retained on the column. By increasing the volumn slightly for each elution step we have increased our yield of labeled aRNA without diluting it too much. ''— [[User:Kam D. Dahlquist\|Kam D. Dahlquist]] 12:47, 22 July 2010 (EDT)''		#* Note: the manufacturer's protocol calls for eluting twice with ''10 μL'' of the nuclease-free water, not 15 μL. However, we have observed that 5-8 μL of the total elution volume is retained on the column. By increasing the volumn slightly for each elution step we have increased our yield of labeled aRNA without diluting it too much. ''— [[User:Kam D. Dahlquist\|Kam D. Dahlquist]] 12:47, 22 July 2010 (EDT)''
	# Let sit at room temperature for 2 minutes, then spin for 3 minutes at 5,000 X g.		# Let sit at room temperature for 2 minutes, then spin for 3 minutes at 5,000 X g.